Abstract: Objective: Comparison of the antibacterial activity of APS with four β-lactam antibiotics against different Staphylococcus aureus (SA) and speculation of the antibacterial mechanism of action of APS by network pharmacological approach. Methods: The MICs of APS and β-lactam antibiotics on MSSA-4 (non-membrane producing sensitive bacteria), MRSA-6 (non-membrane producing resistant bacteria), MSSA-11 (membrane producing sensitive bacteria) and MRSA-12 (membrane producing resistant bacteria) were determined by serial dilution method. The effects of drug resistance and biofilm on MIC were compared by one-way multivariate ANOVA. The structure of APS compound was obtained from PubChem database, and target fishing was carried out using PharmMapper database, and the screened targets were imported into STRING database to establish PPI network diagram and capture node information to establish “compound-target-target interaction” network. Metascape platform was used to perform GO enrichment analysis and KEGG pathway enrichment analysis on key targets to predict their antibacterial mechanism of action. Results: The MIC of MSSA-4, MRSA-6, MSSA-11 and MRSA-12 by APS were 125, 125, 62.5 and 62.5 μg/mL, respectively. Analysis of variance showed that bacterial drug resistance and biofilm had little effect on the antibacterial effect of APS, and had better antibacterial activity against membrane producing bacteria. And four β-Lactam antibiotics were easily affected by bacterial drug resistance and biofilm, resulting in the decrease of the sensitivity of the tested bacteria to antibiotics. 123 potential antimicrobial action targets of APS were obtained by network pharmacology, and the protein interaction network had suggested that ALB, AKT1, MMP9, MAPK1, CASP3, IGF1, MAPK8, HRAS, BCL2L1, ESR1 might be its core targets for the biological functions involved mainly include bacterial response, bacterial adhesion regulation, protein domain specific binding and so on, and mainly acted on focal adhesion, amino sugar and nucleotide sugar metabolism, drug metabolism and other pathways. Conclusion: Bacterial resistance and biofilm did not affect the antibacterial effect of APS. APS had the characteristics of multi-target and multi-pathway to affect the bacterial biofilm and exert antibacterial effects.